When challenged by hypoxia, cells increase the expression of proteins involved in the physiological adaptation to low oxygen environments. This compensatory response depends on the activation of heterodimeric transcription factors, designated hypoxia-inducible factors (HIF).1-3 Three different HIF-alpha subunits (HIF-1α, HIF-2α or HIF-3α) and one beta-subunit (HIF-1β or Aryl Hydrocarbon Receptor Nuclear Translocator, ARNT), all belonging to the basic helix-loop-helix family, have been identified to date.4-6 The beta-subunit is constitutively expressed, while the alpha-subunits are tightly regulated by oxygen levels.7 In normoxia, interaction with the von Hippel-Lindau (VHL) tumor suppressor, a component of the E3 ubiquitin ligase complex, leads to ubiquitinization and subsequent proteasome-dependent degradation of HIF-1α subunits.8-10 In hypoxia, however, HIF-1α subunits lose their ability to interact with VHL, and are subsequently stabilized.7,11 
The nature of the oxygen sensors regulating the VHL-HIF-1α interaction and, therefore, HIF-1α-activity has long remained elusive. Recent evidence indicated that a prolyl hydroxylase (PH) enzyme is involved in oxygen sensing.12, 13 VHL interacts with two independent sites of the HIF-1α subunits containing specific proline residues. Hydroxylation of these proline residues, which is required for the interaction of VHL with HIF-1α,14 is catalyzed by a novel family of mammalian proline hydroxylases named PHD1, PHD2 and PHD3, all belonging to the iron (II)-2-oxoglutarate-dependent dioxygenases family. 15, 16 PHD-dependent hydroxylation of HIF-1α subunits requires oxygen. Thus, under hypoxic conditions, HIF-1α is not hydroxylated, and escapes VHL-dependent degradation. Consequently, HIF-1α subunits are enabled to translocate to the nucleus, where they associate with ARNT to form heterodimers that bind to the DNA-specific HRE (hypoxia response elements) of different target genes.17 Knock-down of PHD-activity enhances the HIF-1α-dependent compensatory gene expression program in vitro,18, 19 and peptides containing the PHD prolyl hydroxylation sites act as competitive inhibitors of endogenous prolyl hydroxylation, causing HIF stabilization and increased vessel growth.20,21 
Nonetheless, it remains to be determined to which extent the PHD enzymes have overlapping or unique tissue-specific activities in vivo, which molecules are downstream PHD targets, and whether specific prolyl hydroxylases could serve as potential drug targets to modulate tissue responses to oxygen deprivation. As hypoxia plays a significant role in ischemic cardiovascular disease, cancer, stroke, and chronic lung disease, understanding the role of each PHD enzyme is of critical importance. Prolyl hydroxylases and non-specific inhibitors have been described in US20040254215, Fibrogen Inc., and in US20040146964, Isis Innovation. As described herein, we report the inactivation of the PHD1, PHD2 and PHD3 locus, as well as inheritance and viability of mice lacking each single PHD. Absence of PHD2 results in embryonic lethality, precluding the analysis of its role in pathological conditions in adult mice.